1. Technical Field
The present invention relates to methods and apparatus for determining a microorganism's sensitivity to an antibiotic in general, and to methods and apparatus for determining the minimum inhibitory concentration of an antibiotic relative to a microorganism in particular.
2. Background Information
The determination of the minimum inhibitory concentration (MIC) of an antibiotic is an essential laboratory test to determine the sensitivity of a microorganism, usually a bacterium, to specific antibiotics. The MIC refers to the minimum concentration of an antibiotic necessary to prevent the microorganism from growing. The type and dose of antibiotics is often predicated upon this type of test, making rapid and accurate results critical to both patient care and cost-effective treatment. Antibiotic sensitivity testing is most commonly performed using the qualitative Kirby-Bauer plate method, but for a quantitative MIC analysis, the tube dilution is most commonly used.
The Kirby-Bauer test utilizes a plate covered with a uniform layer of microbiological growth medium specifically formulated for the test at hand. A number of disks are placed on the layer of growth medium, each containing a specific concentration of an antibiotic being evaluated. Bacteria grows on the medium forming a visible coating, except in the area (generally referred to as the "clear.sub.-- zone") around those disks having sufficient antibiotic concentration to inhibit bacterial growth. The size of the clear zone surrounding a disk is indicative of the microorganism's sensitivity to the antibiotic contained in that particular disk; i.e., the larger the clear zone, the greater the microorganism's sensitivity to the antibiotic contained in the disk. The Kirby-Bauer test is popular because of its simplicity and its ability to evaluate multiple antibiotics at once. A disadvantage of the Kirby-Bauer test is that there are a number of variables which affect the antibiotic concentration at any given point in the growth medium, and thus do not allow a MIC to be calculated. Formulae have been published for calculating the approximate MIC based upon the clear zone size, but these formulae are rarely used and are considered to be approximations at best.
The tube dilution method involves placing an equal amount of target microorganism in a plurality of wells (referred to as "tubes") disposed in a platter, and adding different concentrations of an antibiotic to each tube. The lowest concentration of 5antibiotic in which the target microorganism will not grow determines the MIC for that particular microorganism. A disadvantage of the tube dilution method is that its accuracy depends on the step size in concentration change between tubes. A small step size yields greater accuracy, but may require an impractical number of tubes and effort. In addition, preparing accurate dilutions is an expensive process that increases in cost with the number of tubes. Hence, increasing the accuracy of this method can also increase the cost and time required.
An alternative means of performing a MIC determination is described in U.S. Pat. No. 4,778,758 and others, which involves the use of an "E-Strip", which is a strip that incorporates a precisely formed gradient of a single antibiotic. Calibration marks are disposed along a side of the strip, corresponding to the exact concentration of the antibiotic at that point. The strip is placed onto an inoculated Kirby-Bauer plate and after incubation a clear area will form contiguous with an area of microorganism growth, provided an antibiotic concentration within the gradient exceeds the MIC. The calibration markings corresponding to the border between the clear area and the growth area give the MIC value for the antibiotic being evaluated. Several disadvantages are associated with this method for determining a MIC of an antibiotic including, but not limited to: 1) the strip is difficult to manufacture and consequently expensive; 2) the size of the strip makes it impractical for concurrent multiple antibiotic tests in a single apparatus; and 3) the preparation must be read after a precise period of incubation to achieve optimum accuracy.
U.S. Pat. No. 5,702,684 discloses a method for monitoring antibiotic levels for determining when the antibiotics should be replenished in an industrial plumbing system using a fluorescent marker. That method, however, does not allow the determination of a MIC or any type of antibiotic sensitivity measurement.
What is needed is a method for determining the MIC of an antibiotic for a target microorganism, a method that can determine the MIC in a minimum amount of time, a method that provides an accurate MIC, a method that can simultaneously determine the MIC's of several antibiotics for a target microorganism, and a method that is cost effective.